An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ϳ71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic , respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme. P lant cell walls consist of a heterogeneous mixture of polymers, including cellulose, hemicellulose, pectin, and lignin. In addition to these main components, hydroxycinnamates play a key role in the formation of a complex network of these polymers. While hydroxycinnamates covalently cross-link plant cell wall polysaccharides to each other by ester bonds, links between polysaccharides and lignin are formed mainly by ether linkages (1). Thus, these phenolic acids (mainly ferulic, p-coumaric, and caffeic acids) not only influence the rigidity and mechanical properties of the cell wall but also play a role in plant defense (2). They are linked differently to sugar residues in plants: in grasses such as wheat, barley, or maize, ferulates and p-coumarates are mainly found esterified to the O-5 position of arabinose residues, whereas in pectin of dicotyledons, such as spinach or sugar beet, ferulic acid is esterified to the O-2 position of arabinose and the O-6 position of galactose residues (3). Furthermore, hydroxycinnamates also exist as soluble ester conjugates of quinic acid, the best-known example being chlorogenic acid [3-(3,4-dihydroxycinnamoyl)quinic acid] (4). Particularly high concentrations of chlorogenic acid are present in coffee, apple, pear, and potato tuber (4). The health, cosmetic, and pharmaceutical industries are especially interested in hydroxycinnamates due to their anticarcinogenic, anti-inflammatory, and antioxidant properties (5). Consequently, enzymes that are able to release these phenolic compounds are not only essential for the degradation of plant biomass but also of interest for potential food and medical applications of the released products.One group of these enzymes is the feruloyl esterases (FAEs) (EC 3.1.1.73), a subclass of the carboxylic ester hydrolases (EC 3.1.1). They hydrolyze the ester lin...
